Low molecular weight chemical (LMW) allergens are commonly referred to as haptens. Haptens must complex with proteins to be recognized by the immune system. The majority of occupationally related haptens are reactive, electrophilic chemicals, or are metabolized to reactive metabolites that form covalent bonds with nucleophilic centers on proteins. Nonelectrophilic protein binding may occur through disulfide exchange, coordinate covalent binding onto metal ions on metalloproteins or of metal allergens, themselves, to the major histocompatibility complex. Recent chemical reactivity kinetic studies suggest that the rate of protein binding is a major determinant of allergenic potency; however, electrophilic strength does not seem to predict the ability of a hapten to skew the response between Th1 and Th2. Modern proteomic mass spectrometry methods that allow detailed delineation of potential differences in protein binding sites may be valuable in predicting if a chemical will stimulate an immediate or delayed hypersensitivity. Chemical aspects related to both reactivity and protein-specific binding are discussed.

The need for alternatives to animal based skin sensitization testing has spurred research on the use of in-vitro, in silico and in chemico methods. Glutathione and other select peptides have been used to determine the reactivity of electrophilic allergens to nucleophiles, but these methods are inadequate to accurately measure rapid kinetics observed with many chemical sensitizers. A kinetic spectrophotometric assay involving the reactivity of electrophilic sensitizers to nitrobenzenethiol was evaluated. Stopped flow techniques and conventional UV spectrophotometric measurements enabled determination of reaction rates with half-lives ranging from 0.4 ms (benzoquinone) to 46.2 s (ethyl acrylate). Rate constants were measured for 7 extreme, 5 strong, 7 moderate and 4 weak/non-sensitizers. 17 out of the 23 tested chemicals were pseudo-first order and 3 were second order. In 3 out of the 23 chemicals, deviations from first and second order were apparent where the chemicals exhibited complex kinetics whose rates are mixed order. The reaction rates of the electrophiles correlated positively with their EC3 values within the same mechanistic domain. Nonsensitizers such as benzaldehyde, sodium lauryl sulfate and benzocaine did not react with nitrobenzenethiol. Cyclic anhydrides, diones and aromatic aldehydes proved to be false negatives in this assay. The findings from this simple and rapid absorbance model show that for the same mechanistic domain, skin sensitization is driven mainly by electrophilic reactivity. This simple, rapid and inexpensive absorbance based method has great potential for use as a preliminary screening tool for skin allergens.

The formation, reaction dynamics, and detailed kinetics and mechanism of the reaction between nitrous acid and N-acetylpenicillamine (NAP) to produce S-nitroso-N-acetylpenicillamine (SNAP) was studied in acidic medium. The nitrous acid was prepared in situ by the rapid reaction between sodium nitrite and hydrochloric acid. The reaction is first order in nitrite and NAP. It is also first order in acid in pH conditions at or slightly higher than the pK(a) of nitrous acid. In lower pH conditions, the catalytic effect of acid quickly saturates. Higher acid concentrations also induce a faster decomposition rate of the SNAP, thus precluding the quantitative formation of SNAP from HNO2 and NAP. Both HPLC and quadrupole time-of-flight mass spectrometry techniques proved that SNAP was the sole product produced. No nitrosation occurred on the secondary amine center in NAP, and only the thiol group reacted to form the nitrosothiol. Cu(I) ions were found to be effective SNAP-decomposition catalysts. Cu(II) ions had no effect on the stability of SNAP. Ambient oxygen in reaction solutions was found to have no effect on initial rates of formation of SNAP, products obtained, and stability of SNAP. The formation of SNAP occurs through two distinct pathways. One involves the direct reaction of NAP and HNO2 to form SNAP and eliminate water, and the second pathway involved the initial formation of the nitrosyl cation, NO+, which then nitrosates the thiol. The bimolecular rate constant for the reaction of NAP and HNO2 was derived as 2.69 M(-1) s(-1), while that of direct nitrosation by the nitrosyl cation was 3.00 x 10(4) M(-1) s(-1). A simple reaction network made up of four reactions was found to be sufficient in simulating the formation kinetics and acid-induced decomposition of SNAP.

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Diisocyanates (dNCOs) are the most commonly reported cause of chemically induced occupational asthma, but the ultimate antigenic form is unknown. Reactions of the three most common monomeric dNCOs, hexamethylene dNCO (HDI), methylene diphenylisocyanate (MDI), and toluene dNCO (TDI), with cysteine methyl ester (CME) gave the corresponding bis-dithiocarbamates (HDI-CME, TDI-CME, and MDI-CME). The dissociation kinetics of these bis-thiocarbamates, in aqueous conditions, was followed spectrophotometrically under varying pH and temperature conditions. Reaction of the adducts with methylamine or human serum albumin (HSA) produced diurea, monourea, and diamine products, and this was consistent with the base-catalyzed elimination reaction (E1cB) pathway being the dominant, but not exclusive, dissociation mechanism. The hydrolysis of the adducts was first-order with respect to OH(-) concentration and overall second-order (HDI-CME, k = 3.36 x 10(2) M(-)(1) min(-)(1); TDI-CME, k = 2.49 x 10(4) M(-)(1) min(-)(1); and MDI-CME, k = 5.78 x 10(4) M(-)(1) min(-)(1) at pH 7.4) with deviation from second-order when the dNCO had an aromatic functional group. Arrhenius plots gave activation energies (HDI-CME, E(a) = 70.6 kJ/mol; TDI-CME, E(a) = 46.1 kJ/mol; and MDI-CME, E(a) = 44.5 kJ/mol) that were consistent with the following order of stability: HDI-CME > TDI-CME > MDI-CME. Therefore, the stability of different dNCO-derived thiocarbamates in aqueous environments can vary greatly. Thiocarbamate dissociation rates and type of products formed may potentially influence antigenicity and subsequent hypersensitivity/toxic reactions following dNCO exposures.

Protein haptenation by polyurethane industrial intermediate methylene diphenyl diisocyanate (MDI) is thought to be an important step in the development of diisocyanate (dNCO)-specific allergic sensitization; however, MDI haptenated albumins used to screen specific antibody are often poorly characterized. Recently, the need to develop standardized immunoassays using a consistent, well characterized dNCO-haptenated protein to screen for the presence of MDI-specific IgE and IgG from workers’ sera has been emphasized and recognized. This has been challenging to achieve due to the bivalent, electrophilic nature of dNCO leading to the capability to produce multiple cross-linked protein species and polymeric additions to proteins. In the present study, MDI was reacted with human serum albumin (HSA) and hemoglobin (Hb) at molar ratios ranging from 1:1 to 40:1 MDI: protein. Adducts were characterized by (1) loss of available trinitrobenzene sulfonic acid (TNBS) binding to primary amines, (2) electrophoretic migration in polyacrylamide gels, (3) quantification of methylene diphenyl diamine following acid hydrolysis and (4) immunoassay. Concentration dependent changes in all the above noted parameters were observed demonstrating increase in both number and complexity of conjugates formed with increasing MDI concentration. In conclusion, a series of bio-analytical assays should be performed to standardize MDI-antigen preparations across lots and laboratories for measurement of specific antibody in exposed workers which in total indicate degree of intra- and inter-molecular cross-linking, number of dNCO bound, number of different specific binding sites on the protein and degree of immuno-reactivity.

Benzoquinone (BQ) is an extremely potent electrophilic contact allergen that haptenates endogenous proteins through Michael addition (MA). It is also hypothesized that BQ may haptenate proteins via free radical formation. The objective of this study was to assess the inductive effects (activating and deactivating) of substituents on BQ reactivity and mechanistic pathway of covalent binding to a nucleophilic thiol. The BQ binding of Cys34 on human serum albumin was studied and for reactivity studies, nitrobenzenethiol (NBT) was used as a surrogate for protein binding of the BQ and benzoquinone derivatives (BQD). Stopped flow techniques were used to determine pseudo-first order rate constants (k) of methyl, t-butyl and chlorine substituted BQD reactions with NBT whereas electron pair resonance (EPR) studies were performed to investigate the presence a free radical mediated binding mechanism of BQD. Characterization of adducts was performed using mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). The rate constant values demonstrated the chlorine substituted (activated) BQD to be more reactive toward NBT than the methyl and t-butyl substituted (deactivated) BQD, and this correlated with the respective EPR intensities. The EPR signal, however, was quenched in the presence of NBT suggesting MA as the dominant reaction pathway. MS and NMR results confirmed adduct formation to be a result of MA onto the BQ ring with vinylic substitution also occurring for chlorine substituted derivatives. The binding positions on BQ and NBT:BQ(D) stoichiometric ratios were affected by whether the inductive effects of the substituents on the ring were positive or negative. The reactivity of BQ and BQD is discussed in terms of the potential relationship to potential allergenic potency.

The rubber accelerator, 2-mercaptobenzothiazole (MBT), has been reported to cause allergic contact dermatitis from gloves and other rubber products, but its chemical fate when exposed to occupational oxidants and the mechanism of its pathogenesis are not known. It was hypothesized that the thiol group is critical to MBT's (its oxidation products or metabolites) covalent binding and/or haptenation to nucleophilic protein residues. Oxidative transformation of MBT to the disulfide 2,2'-dithiobis(benzothiazole) (MBTS) was observed within the glove matrix when hypochlorous acid, iodine, and hydrogen peroxide were used as oxidants. Cysteine reduced MBTS to MBT with subsequent formation of the mixed disulfide 2-amino-3-(benzothiazol-2-yl disulfanyl)propionic acid which was identified and characterized. Spectrophotometry and mass spectrometry experiments demonstrated the simultaneous reduction of MBTS and disulfide formation with Cys34 on bovine serum albumin, suggesting a potential route of protein haptenation through covalent bonding between protein cysteinyl residues and the MBT/MBTS thiol moiety. Metabolism of MBT using isoniazid and dexamethasone-induced rat liver microsomes, to give a protein reactive epoxide intermediate and provide an alternative protein haptenation mechanism, was not observed. The data suggest that the critical functional group on MBT is the thiol, and haptenation is via the formation of mixed disulfides between the thiol group on MBT and a protein sulfhydryl group.